This is a proposal to develop a method for generating small activating RNAs (saRNAs) that specifically activate genes in mammalian cells. Activating genes is a powerful technique that can be used to develop stem cells in an ethical manner, manipulate cells to study basic cell biology, and develop/test therapeutics. Currently, up-regulation of gene expression in mammalian cells is usually carried out through the introduction of cDNA expression vectors into cells. This is cumbersome and has a low level of efficiency. Alternatively, the newly reported RNAa technique may be a preferable tool for the activation of genes. The use of saRNA has important advantages over the currently used cDNA method including the ability to transduce multiple saRNAs using a single vector and the ability to activate genes with alternatively spliced forms by using a single saRNA species. The RNAa technique is still in its infancy and its general use is limited because there is a learning curve for designing effective saRNAs and developing appropriate vectors for them. For example, it required about 5 years for the field of RNAi to become routine for any standard molecular biology facility to carry out. In order to jump start the general availability and use of saRNA technology, we will develop an HTS method for developing saRNAs. We will use this method to build a commercial inventory of saRNAs to the most popular gene promoters and use the technology to fill custom orders. In this Phase I study, we will validate our HTS methods by developing saRNAs to the promoters of two high visibility pluripotency- inducing genes and two tumor suppressor genes. In Experiment #1 we will generate four large lentiviral libraries expressing candidate saRNA molecules to our four promoters of interest. In Experiment #2 we will test the saRNAs for efficiency in activating their respective promoters. In Experiment #3 we will develop and test a preliminary algorithm for designing additional saRNAs based on our successes and failures in Experiment #2. Our test of feasibility for our approach will be whether or not we have generated saRNAs that specifically activate the four target genes and whether we are able to increase our success rate for developing active saRNAs using the preliminary algorithm that we develop in Experiment 3. PUBLIC HEALTH RELEVANCE: This is a proposal to develop a method for generating small RNAs that specifically activate genes in mammalian cells to facilitate biological research and drug discovery.